M2 macrophage-derived exosomal circTMCO3 acts through miR-515-5p and ITGA8 to enhance malignancy in ovarian cancer

Tumor-associated macrophages of the M2 phenotype promote cancer initiation and progression. Importantly, M2 macrophage-derived exosomes play key roles in the malignancy of cancer cells. Here, we report that circTMCO3 is upregulated in ovarian cancer patients, and its high expression indicates poor survival. M2-derived exosomes promote proliferation, migration, and invasion in ovarian cancer, but these effects are abolished by knockdown of circTMCO3. Furthermore, circTMCO3 functions as a competing endogenous RNA for miR-515-5p to reduce its abundance, thus upregulating ITGA8 in ovarian cancer. miR-515-5p inhibits ovarian cancer malignancy via directly downregulating ITGA8. The decreased oncogenic activity of circTMCO3-silencing exosomes is reversed by miR-515-5p knockdown or ITGA8 overexpression. Exosomal circTMCO3 promotes ovarian cancer progression in nude mice. Thus, M2 macrophage-derived exosomes promote malignancy by delivering circTMCO3 and targeting the miR-515-5p/ITGA8 axis in ovarian cancer. Our findings not only provide mechanistic insights into ovarian cancer progression, but also suggest potential therapeutic targets.

Ovarian cancer is one of the most common and fatal gynecological malignancies 1 .Due to the lack of effective early diagnosis strategies, ovarian cancer is typically diagnosed at an advanced stage with low 5-year survival rate of 29% 2 .Platinum-based chemotherapy, such as cisplatin, is one of standard therapies for ovarian cancer 3,4 .However, cisplatin resistance is one of major causes of therapeutic failure.Therefore, elucidating the mechanisms underlying the malignancy of ovarian cancer is crucial for developing therapeutic strategies.
Macrophages are crucial immune cells that can phagocytize bacteria and infected cells, present antigens and trigger inflammation 5 , which are typically divided into classically activated M1 macrophages and alternatively activated M2 macrophages.Importantly, tumor-associated macrophages (TAMs) account for about 50% of tumor mass with M2-polarized phenotypes 6,7 .TAMs are abundant in patients with ovarian cancer 8 , and TAMs promote ovarian cancer progression via regulating immune escape, migration, invasion, and metastasis 9 .However, TAM-mediated regulation of malignancy of ovarian cancer cells remains largely unknown.One of important mechanisms for macrophages is to release exosomes 10 .Exosomes released by M2 macrophages enhance migration and invasion in colon cancer 11 .Exploring macrophage-derived exosome-mediated regulation of ovarian cancer is crucial for a better understanding of the pathogenesis of ovarian cancer.
Circular RNAs (circRNAs) are non-coding RNAs which are formed by back-splicing 12 and enriched in exosomes 13 .Strikingly, emerging evidence has revealed key roles of exosomal circRNAs in various human cancers.Chen and colleagues found that exosome-derived circ_0051443 repressed the malignancy of hepatocellular carcinoma (HCC) cells and HCC progression by promoting BAK1 expression 14 .In ovarian cancer, exosomal circWHSC1 promoted MUC1 expression and peritoneal diffusion and adhesion, contributing to cancer metastasis 15 .Guan et al. found that cir-cPUM1 facilitated ovarian cancer progression, and exosomal circPUM1 promoted metastasis 16 .Thus, understanding the mechanisms by which exosomal circRNAs regulate ovarian cancer progression is truly important.A previous study reported increased abundance of circ_0031017 derived from TMCO3 gene (also known as circTMCO3) in TAM-derived exosomes 17 , suggesting the potential roles of exosomal circ_0031017 derived from M2 macrophages in cancers.Additionally, circTMCO3 was upregulated, and it sponged miR-577 to enhance proliferation, migration, and invasion in gastric cancer 18 .Wang et al. reported that circTMCO3 was highly expressed in HCC, and circTMCO3 might represent potentially valuable diagnostic biomarkers for HCC 19 .However, to our knowledge, the roles of circTMCO3 in cancers remain largely unknown, especially in ovarian cancer.Therefore, we focused the function of M2-derived exosomal circTMCO3 in ovarian cancer.
One of action mechanisms of circRNAs is that circRNAs function as competitive endogenous RNAs (ceRNAs) to absorb microRNAs (miRNAs) and release inhibitory effects on downstream targets 20 .CircRNAs modulate tumor-related gene expression via sponging miRNAs.miR-515-5p works as a tumor suppressor to inhibit proliferation, migration, and metastasis 21 .Integrins are key regulators of cancer growth and metastasis 22 .Integrin subunit alpha 8 (ITGA8) promotes epithelial-mesenchymal transition and cell invasion in multiple myeloma 23 .However, their roles and interaction in ovarian cancer are unknown.We found that miR-515-5p had a targeted binding relationship with circTMCO3 and ITGA8, respectively.Therefore, we hypothesized that M2-derived exosomal circTMCO3 might regulate malignant behaviors in ovarian cancer via sponging miR-515-5p and upregulating ITGA8.
To summarize, our investigation aims to study the roles of M2 macrophage-derived exosomes carrying circTMCO3 in regulating the malignancy of ovarian cancer cells.We found that the increased circTMCO3 expression was associated the poor survival of patients with ovarian cancer.Further investigation demonstrated that M2 macrophage-derived exosomal circTMCO3 promoted the malignancy by targeting the miR-515-5p/ITGA8 axis in ovarian cancer.Our findings are beneficial for deepening understanding of the progression of ovarian cancer and providing potential exosome-based therapeutic strategies.

Abundant M2 macrophages facilitate ovarian cancer cell proliferation, migration, and invasion
To investigate macrophage-mediated regulation of tumor progression, ovarian cancer tissues from patients were collected, and the M2 macrophage marker CD206 was detected via immunohistochemistry (IHC) staining.Compared to normal tissues, ovarian cancer tissues showed increased CD206-positive cells, suggesting increased abundance of M2 macrophages in ovarian cancer tissues (Fig. 1a).NTMs and TAMs were isolated, and the ratio of M2 macrophages (CD163 and CD206 double positive) was significantly increased in TAMs (Fig. 1b).Macrophages were isolated from tumor (TAM) and normal (NTM) tissues.Compared to NTMs, TAMs exhibited decreased the expression of M1 macrophagerelated factors TNF-α and iNOS but increased the expression of M2 macrophage-associated factors Arg-1 and IL-10 (Fig. 1c).These data demonstrated that M2 macrophages were highly abundant in ovarian cancer.To explore whether M2 macrophages regulate the malignant behaviors of ovarian cancer cells, THP-1 cells were polarized to M2 macrophages, and A2780 and SKOV3 cells were cocultured with M0 or M2 macrophages.GW4869 was added to inhibit exosome generation.GW4869 significantly reduced total exosomal protein from M2 macrophages (Supplementary Fig. 1).Coculture with M2 macrophages greatly enhanced tumor cell proliferation, but it was abrogated by GW4869 (Fig. 1d, e).Furthermore, cell migration and invasion were enhanced by M2 macrophages, but these effects were largely suppressed by GW4869 treatment (Fig. 1f, g).These results suggest that abundant M2 macrophages promote malignancy in ovarian cancer cells via secreting exosomes.
CircTMCO3 is upregulated in ovarian cancer and M2 macrophage-derived exosomes can be internalized by ovarian cancer cells We isolated exosomes from TAMs and NTMs and examined the expression of hsa_circ_0000772, hsa_circ_0084335, hsa_circ_0031017 (circTMCO3), hsa_circ_0062531, hsa_circ_0017992, hsa_circ_0091577, and hsa_circ_0065492 in TAMs, NTMs and exosomes.hsa_circ_0000772, hsa_circ_0084335, hsa_circ_0031017 (circTMCO3) and hsa_circ_0091577 were upregulated in both TAMs and exosomes from TAMs, and hsa_circ_0031017 showed highest expression in TAMs (Supplementary Fig. 2a).Moreover, we examined the expression of cir-cTMCO3 in THP-1 cells, M0 and M2 macrophages and found that cir-cTMCO3 was highly expressed in M2 macrophages derived from THP-1 cells (Supplementary Fig. 2b).In addition, Northern blot assays showed that, compared to exosomes derived from NTMs, THP-1 cells and M0 macrophages, exosomes from TAMs and M2 macrophages derived from THP-1 cells showed increased circTMCO3 expression, respectively (Supplementary Fig. 2c).As circTMCO3 was abundant in cancer and TAM-derived exosomes, we analyzed circTMCO3 expression in ovarian cancer tissues from patients.CircTMCO3 expression was increased, and patients with high circTMCO3 expression exhibited poor survival (Fig. 2a, b).Strikingly, coculture with M2 macrophages upregulated circTMCO3 in A2780 and SKOV3 cells, and GW4869 treatment blocked circTMCO3 upregulation (Fig. 2c), indicating that M2 macrophages might enhance circTMCO3 expression through exosomes.As illustrated in Fig. 2d, circTMCO3 is generated from the exons 2-8 of TMCO3 on chromosome 13 with a length of 1423 nucleotides (nt) through backsplicing, and the junction site was verified by Sanger sequencing.Circular RNAs are quite stable in response to actinomycin D and RNase R treatment 24,25 .We found that, compared to linear TMCO3 mRNA, cir-cTMCO3 exhibited decreased RNA decay after actinomycin treatment and high resistance to RNase R digestion (Fig. 2e, f).In addition, nuclear and cytoplastic fractions were prepared, and circTMCO3 could be detected in both nuclear and cytoplastic fractions, but it primarily localized in the cytoplasm (Fig. 2g).GW4869 suppressed circTMCO3 expression and M2-meditated oncogenic activity, suggesting that exosomes generated by M2 macrophages might be implicated in regulating circTMCO3 expression and ovarian cancer progression.Exosomes were then examined by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA).Distinctive round bilayer vesicles with mainly 30-150 nm in diameter were observed (Fig. 2h, i).Moreover, M2 macrophage-derived exosomes showed high levels of extracellular vesicle proteins including tetraspanins (CD63, CD9, CD81) and TSG101 26 compared to M2 macrophages (Fig. 2j).In coculture assays, we found that PKH26-labeled exosomes were internalized by ovarian cancer cells (Fig. 2k).The ratio of PKH26-positive cells was ~30% at 12 h, and it was increased to ~65% at 24 and 48 h (Fig. 2k).These results suggest that M2 macrophages raise circTMCO3 level in cancer cells via exosomes.

Discussion
Ovarian cancer is a highly lethal cancer that accounts for 5% of all cancerrelated deaths among women 31,32 .Ovarian cancer is generally diagnosed at a late stage, causing poor outcome of patients 33 .Therefore, exploring the regulatory mechanisms of ovarian cancer progression is very important.Here, we firstly demonstrated that M2 macrophage-derived exosomal  circTMCO3 sponged miR-515-5p and relieved its inhibitor effect on ITGA8 expression, thereby promoting the malignancy and progression of ovarian cancer.
Exosomes play vital roles in cell-cell communication via transferring cell components and regulating behaviors of various cancer cells 34,35 .M2 macrophages attract much attention as the major component of tumor microenvironment.Exosomes released by M2 macrophages promote the metastasis of hepatocellular carcinoma cells and accelerate angiogenesis and growth of pancreatic cancer 36,37 .In consistence, we observed that M2 macrophages enhanced ovarian cancer cell proliferation, migration, and invasion via releasing exosomes.Exosomes have shown enormous potential in drug delivery with great biocompatibility and low immunogenicity 38 .Our data suggests that M2 macrophage-derived exosomes can be engineered for the management of ovarian cancer.
In recent years, exosomes delivering ncRNAs, such as lncRNAs, are becoming a pivotal regulator in regulating cancer progression 39 .Exosomal circRNAs have been regarded as potential therapeutic targets for various cancers 40 .Ma et al. found that exosomal circRNA051239 derived from tumors promoted proliferation, migration, and invasion of epithelial ovarian cancer cells via working as a miR-509-5p sponge and regulating PRSS3 expression 41 .In addition, circRNAs are key regulators of ovarian cancer progression and therapeutic targets for ovarian cancer 42 .However, our understanding of circRNAs and related regulatory mechanisms are still limited.CircTMCO3 exerts an oncogenic activity in gastric cancer, but its roles in other cancers including ovarian cancer remain unknown.Here, increased expression of circTMCO3 in M2 macrophages suggested us that exosomes might carry circTMCO3 to exert its function in ovarian cancer.Indeed, exosomes with knockdown of circTMCO3 lost their activity in regulating malignancy, demonstrating that exosomes exert their function via delivering circTMCO3 in ovarian cancer.
The ceRNA hypothesis suggests that lncRNAs work as miRNA sponges to upregulate downstream mRNA targets in cancers 43 .miR-577 has been identified as a target of circTMCO3 18 .Wen et al. discovered that miR-515-5p repressed malignant behaviors in breast cancer through CBX4 44 .miR-515-5p restrained migration and metastasis by downregulating MARK4 in lung and breast cancers 45 .However, the implication of miR-515-5p in ovarian cancer is unknown.Here, miR-515-5p was identified as a target of circTMCO3, and miR-515-5p restrained proliferation, migration, invasion, and EMT, discovering an anti-tumor activity in ovarian cancer.
The integrin family serves key roles in promoting cancer progression via facilitating proliferation, migration, invasion, and metastasis in various cancers 46 .As an integrin subunit, ITGA8 promotes EMT, migration, and invasion in multiple myeloma 23 , and its diagnostic and prognostic values in colon cancer have been validated 47 .However, its roles in ovarian cancer have never been reported previously.We showed ITGA8 as a downstream target of miR-515-5p in ovarian cancer.Our findings suggested that miR-515-5p reduced ITGA8 expression via binding to its 3′ UTR in ovarian cancer, and ITGA8 overexpression reversed miR-515-5p-mediated regulation of cancer malignancy and progression, indicating that miR-515-5p reduced ITGA8 expression to suppress proliferation, migration, invasion, and metastasis.However, ITGA8 is transmembrane receptor that mediates numerous cellular processes, and its implication in exosome internalization needs to be explored.Besides regulating ITGA8 expression, exosomal RNA-mediated regulation of ITGA8 function might be implicated in the pathogenesis of ovarian cancer.Taken together, M2 macrophage-derived exosomes enhance malignancy in ovarian cancer by delivering circTMCO3 and targeting the miR-515-5p/ITGA8 axis.Our findings highlight exosomal circTMCO3-mediated regulation of malignancy of cancer cells and provide potential therapeutic strategies for ovarian cancer.Single cell sequencing will be performed in future studies to investigate the gene expression level difference between the cell types within the tumor and normal tissues.

Methods
Patients and specimens Sixty patients were diagnosed with ovarian cancer at Hunan Cancer Hospital, and tumor and adjacent normal tissues were collected and stored for RNA extraction and immunohistochemistry staining.Inclusion criteria was following: 1 Greater than or equal to 18 years old; 2 First diagnosis with primary ovarian cancer; 3 No other severe diseases, such as cancers and diabetes; 4 No treatment; 5 No pregnancy and lactation.Macrophages were isolated from tumor (TAM) and normal (NTM) tissues and stained with CD11b and CD206 antibodies (101205 and 321105, Biolegend, San Diego, CA, USA).The cells were detected Beckman cytoflex flow cytometry (Beckman, USA), and the Cytexpert Software was applied for the flow cytometry data analysis.The gating strategy was provided in the Supplementary Fig. 4. Written informed consent was required from patients.The Ethics Committee of Hunan Cancer Hospital approved this study.

Exosome extraction, characterization, and internalization
Exosomes were extracted from M2 macrophages using Total Exosome Isolation Reagent (Invitrogen).Culture media were centrifuged, and the supernatants were collected.The exosome isolation reagent was mixed with the supernatants thoroughly and incubated overnight at 4 °C.Samples were centrifuged, and the pellet was resuspended.Exosomes were quantified and co-cultured with A2780 or SKOV3 cells at 100 µg/mL for 48 h.TEM was applied to examine exosome morphology.exosome size was analyzed by NTA (Malvern, Westborough, MA, USA).Protein levels of CD9, CD63, CD81 and TSG101 were determined by western blotting.Exosomes were stained with PKH26 dye (Sigma) and incubated with A2780 and SKOV3 cells for 24 h for imaging.
CircTMCO3 characterization SKOV3 cells were treated with actinomycin D (5 µg/mL, Sigma) for 0, 4, 8, 12, or 24 h, and RNA was extracted for analyzing the decay of circTMCO3 and TMCO3 mRNA.Moreover, RNA was isolated, treated with RNase R (5 U/µg, Abcam, Cambridge, UK) for 2 h and subjected RT-qPCR for examining the remaining of circTMCO3 and TMCO3 mRNA.CircTMCO3 was amplified using divergent primers, and the junction site was identified through Sanger sequencing (Sangon Biotech, Shanghai, China).

Nuclear and cytoplasmic fractionation
Nuclear/Cytosol Fractionation Kit was purchased from BioVision (Milpitas, CA, USA).Nuclear and cytoplasmic fractions were separated following the manual.The abundance of circTMCO3, U6 snRNA and GAPDH in nuclear and cytoplasmic fractions was examined by RT-qPCR.
Fluorescence in situ hybridization (FISH) SKOV3 cells were seeded on coverslips and fixed in a mixture solution of methanol and acetic acid (3:1) for 10 min.Let coverslips dried naturally, and cells were immersed in hybridization solution containing Alexa Fluor 488labeled circTMCO3 and Cy3-labeled miR-515-5p probes at 25 nM.Coverslips were denatured and incubated overnight.Coverslips were washed and mounted in antifade mountant with DAPI (Beyotime, Shanghai, China) for imaging.

Northern blotting
Cells and exosomes were lysed, and total RNA was extracted and quantified by measuring the OD at 260 nm.Subsequently, RNA was separated by agarose gel electrophoresis and transferred to Nylon membrane.RNA was cross-linked to the membrane, prehybridized, and hybridized with the radiolabeled circTMCO3 probe.Then, the membrane was washed, and the signal was visualized using X-ray 27 .

Wound healing assay
In brief, inserts were oriented in plates.A2780 and SKOV3 cells (5 × 10 5 ) were added and cultured at 37 °C.Then, inserts were removed, and cells were washed and cocultured with macrophages or exosomes as indicated.The wound healing process was monitored using the BX51 microscope from Olympus (Tokyo, Japan).
EdU incorporation assay A2780 and SKOV3 cells were cocultured with exosomes as indicated in medium supplemented with EdU (10 µM, Thermo Fisher Scientific) for 24 h.Cells were fixed, permeabilized and processed for the Click-IT reaction following the manual.Cells were stained with DAPI (Beyotime) and captured using a confocal microscope (Nikon).The EdU positive rate (%) = the number of positive cells/total cells ×100%.

Transwell invasion assay
Cell invasion was assessed by transwell assays with transwell chambers (8µm pore membranes, Corning, NY, USA) precoated with Matrigel (Corning).Cells (1 × 10 6 cells) were seeded on the upper chamber and cocultured with macrophages or exosomes as indicated.Cells which invaded into the lower chamber were fixed, stained with crystal violet (Sigma) and observed under the BX51 microscope (Olympus).

RNA immunoprecipitation (RIP)
RIP assays were performed using the RIP kit (Millipore, Burlington, MA, USA).Magnetic beads were coated with the rabbit anti-Ago2 (ab186733, Abcam) or normal IgG (2729, Cell Signaling Technology, Danvers, MA, USA).A2780 and SKOV3 cells were lysed, and lysates were harvested.Magnetic beads and lysates were mixed well and incubated overnight.RNA was recovered and subjected to RT-qPCR.
RNA pull-down A2780 and SKOV3 cells were lysed and centrifugated for collecting cell lysates.Biotinylated circTMCO3 probe or control oligo probe was mixed with cell lysates and incubated for 16 h at 4 °C.Streptavidin magnetic beads (Thermo Fisher Scientific) were added, mixed well, and incubated for additional 2 h with gentle rotation.Subsequently, RNA was recovered, and the abundance of miR-515-5p was examined by RT-qPCR.

An intraperitoneal xenograft mouse model of ovarian cancer
Twenty female BALB/c nude mice (five-week-old, Hunan SJA Laboratory Animal, Changsha, China) were randomly divided into four groups (n = 5 per group): Control, Exo, Exo-shNC, Exo-shcircTMCO3#1 and Exo-shcircTMCO3#2.SKOV3 cells (1 × 10 6 cells in 200 µL of PBS) were injected intraperitoneally into nude mice.Subsequently, exosomes (30 µg in 200 µL of PBS) from M2, shNC or shcircTMCO3-transfected M2 macrophages were intraperitoneally injected into mice every three days.Control mice received 200 µL of PBS without exosomes.Mice were sacrificed through deep anesthesia with sodium pentobarbital after 21 days.The investigator was blinded to the group allocation during the experiment.Animal procedures were approved by the Animal Care and Use Committee of Hunan Cancer Hospital.

Statistics and reproducibility
Data from three independent experiments were presented as mean ± standard deviation.Kaplan-Meier curve was applied to analyze patient survival, and the survival time was statistically compared using the log rank test.The Pearson correlation was used to for correlation analysis of gene expression.A priori power analysis (G*Power software) was performed to assess sample size required to generate 80% power for detecting a significant (P < 0.05) effect of treatment.The normality of data was evaluated by the Shapiro-Wilk test.Considering the significance level of 5%, there were no significant deviations from the normality of all data (P > 0.05).The variance of groups was analyzed by the Student's t test (two tailed) and one-way analysis of variance (ANOVA) with Turkey post hoc test.P < 0.05 was statistically significant.*P < 0.05, **P < 0.01 and ***P < 0.001.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 2 |
Fig. 2 | CircTMCO3 was upregulated and M2 macrophage-derived exosomes could enter ovarian cancer cells.a CircTMCO3 expression was determined by RT-qPCR in patient tissues.b Patient survival.c RT-qPCR analysis of circTMCO3 in A2780 and SKOV3 cells in coculture assays (n = 3).d Genomic loci and the junction site of circTMCO3.Sanger sequencing was applied to confirm the junction site.e, f The abundance of circTMCO3 and linear TMCO3 mRNA in SKOV3 in response to actinomycin D and RNase R treatment was determined through RT-qPCR (n = 3).g The abundance of circTMCO3, U6 and GAPDH in nuclear and cytoplasmic fractions from SKOV3 cells was examined by RT-qPCR (n = 3).h The examination of M2 macrophage-derived exosomes using TEM.Scale bar: 200 nm.i Exosome size was evaluated by NTA.j Protein levels of CD63, CD9, CD81 and TSG101 in M2 macrophages and exosomes were analyzed by western blotting.k Exosomes (Red) were labeled with PKH26 and endocytosed by A2780 and SKOV3 cells (Nuclei, blue) after 12, 24 and 48 h.Scale bar: 25 μm.*P < 0.05, **P < 0.01 and ***P < 0.001.Data were presented as mean ± standard deviation.